Clin. exp. Immunol. (1976) 26, 371-374.
Specific antibody production against a soluble antigen in the Harderian gland of the domestic fowl R. B. BURNS Anatomy Section, A.R.C. Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh
(Received 22 June 1976) SUMMARY
Three groups of chickens were immunized with bovine serum albumen (BSA). One group received the antigen i.v., the second had BSA dropped into the orbit and the third group was immunized both systemically and by topical application of BSA. Agar-gel diffusion studies showed antibodies to BSA to be present in the tears and sera of the first and third groups but only in the tears of the second group. Immunofluorescence revealed positive anti-BSA staining in lymphoid cells of the spleens of the first and third groups and in plasma cells of Harderian glands of all three groups. It is concluded that systemic and/or topical applications of BSA affect the tears and Harderian gland whereas only systemic injections produce an effect in the spleen and serum. The Harderian gland may have an important part to play in the local immunologic mechanism of the eye and upper respiratory tract of the fowl.
INTRODUCTION Harder's gland is the dominant orbital gland in the domestic fowl (Wight et al., 1971). Though its main functions are to lubricate the movement of the nictitating membrane, wet the surface of the eyeball and nourish the avascular cornea, it probably plays a part in the local immunity of the eye and upper respiratory tract since it has a large age-dependent population of plasma cells (Bang & Bang, 1968; Wight et aL, 1971), surface immunoglobulin-bearing cells (Albini & Wick, 1973) and some antibodyproducing cells (Mueller, Sato & Glick, 1971). Sundick, Albini & Wick (1973) have shown that the lymphoid cells in the gland are mainly bursa-dependent suggesting that the gland is active in humoral, as opposed to cellular, antibody responses (Cooper et al., 1966). The present experiments study the effects of conjunctival and systemic immunizations with bovine serum albumen on the Harderian gland and tears of the domestic fowl.
MATERIALS AND METHODS Animals. Nine-week-old White Leghorn chickens were used. Immunization. Eighteen birds were divided into three groups of six. Group A birds received an i.v. injection of 0 5 ml 300 bovine serum albumen (BSA) in saline twice a week for 2 weeks. Group B received eye drop instillations (0.5 ml into each eye) of 3%o BSA twice a week for two weeks and group C received similar amounts of 3% BSA both i.v. and dropped into
the orbit twice a week for 2 weeks. Untreated and saline-treated birds served as controls. A week after the final injections and orbital applications of BSA the birds were bled from the brachial vein to obtain serum and tears were collected. Tear collection. Lachrymation was induced by dropping glycerol onto the eyeball. This immediately caused tears to well up into the pocket formed by gently pinching the lower eyelid. About 0 5 ml of fluid was collected from each eye using a Pasteur pipette. About two thirds of the fluid collected, as estimated by subtracting the glycerol used from the total volume of fluid, appeared to be composed of tears. Ouchterlony Plate Analyses. Antibodies against BSA were detected by double diffusion in agar gel (Ionagar No. 2, Oxoid) using 8%NaCl in the agar (Aitken & Parry, 1974). Neat serum and undiluted tear-glycerol mixtures were used in the
1%y
Correspondence: Mr R. B. Burns, Mains Road, Edinburgh EH9 8JF.
Agricultural Research Council's Poultry Research Centre, King's Buildings, West
371
372
R. B. Burns
peripheral wells and 1% BSA in phosphate-buffered saline in the central well. Agar plates were stained with naphthalene black. Immunofluorescence. Immediately after the blood and tear collections the birds were killed with pentobarbitone sodium ('Nembutal', Abbott Laboratories). The Harderian glands and spleen were removed, fixed in cold 95% ethanol and waxembedded according to the method of Sainte-Marie (1963). Wax sections, 4 ,um thick, were cut, mounted on glass slides and stained for specific histochemical demonstration of antibody using the method of Coons, Leduc & Connolly (1955). Dewaxed sections were flooded with 1% BSA in saline and incubated at room temperature for 30 min in a humid atmosphere. After washing in phosphate-buffered saline (PBS) the sections were incubated with fluorescein-conjugated rabbit anti-.BSA antiserum (Nordic Pharmaceuticals) diluted 1:10 with PBS for 30 min. The sections were again washed in PBS and then mounted in buffered glycerol. Control sections were prepared in a similar manner except that the antigen layer was omitted. Preparations were examined using a microscope fitted with a mercury vapour lamp. Fluorescence photographs were taken on Ilford HPA film with a constant exposure of 60 sec.
RESULTS The presence of antibody against BSA in tears and serum was demonstrated by agar-gel diffusion. Table 1 summarizes the results. The serum from all six birds in group A showed a positive reaction against BSA in the agar plates whereas only three out of six birds had antibodies against BSA in their tears. Four out of six birds in group B had antibodies against BSA in their tears though none of the sera in this group showed a positive reaction against BSA. The results of group C tears and sera were similar to those of group A with all six sera having antibodies against BSA and four out of the six tear collections being positive. When serum reacted against BSA in Ouchterlony plates a single precipitin line was formed (Fig. 1) whereas when tears showed antibodies against BSA two precipitin lines formed (Fig. 1). Because the TABLE 1. Ouchterlony plate analyses of sera and tears from BSA-stimulated birds
Group A Group B Group C
Number of birds
Number of birds
Route of immunization
showing positive sera
showing positive tears
i.v. Injections Ocular drops i.v. Injections and ocular drops
6/6 0/6 6/6
3/6 4/6 4/6
FiG. 1. Double-diffusion test in agar gel. Bovine serum albumen (BSA) in the centre well with serum (S) and tears (T) from a bird immunized by eyedrop and i.v. injection of BSA in the side wells. Stained with naphthalene black.
Fowl Harderian gland antibody production 373 inner of the two precipitin lines produced by the tears gave a reaction of identity with the single line given by the serum it is suggested that in the serum an IgG response has taken place whereas in the tears both IgG and IgA are present as antibodies. When sections of Harderian gland and spleen were stained by the sandwich technique for anti-BSA fluorescence, anti-BSA activity was seen in the glands of all three experimental groups. Fluorescence was confined to plasma cells (Fig. 2). It was usual for the whole cytoplasm of the cell to fluoresce a bright apple-green colour (Fig. 2) though occasionally plasma cells with brightly fluorescing spots (immunoglobulin surface determinants) were also seen (Fig. 3).
FIG. 2. Harderian gland plasma cells showing specific fluorescence for anti-BSA antibody in a bird immunized by eye-drop applications of BSA. (Magnification x 384.)
FIG. 3. Harderian gland plasma cells (arrowed) showing fluorescent spots specific for anti-BSA antibody in a bird immunized by eyedrop and i.v. injection of BSA. (Magnification x 384.)
Anti-BSA fluorescing plasma cells in the Harderian glands occurred either singly or in groups of four or five (Figs 2 and 3). Occasionally the tracts between epithelial cells lining the primary and secondary tubules showed some specific fluorescence. Positive anti-BSA fluorescence was seen in lymphoid cells of spleens of group A and C birds but not in group B. Control sections of Harderian gland and spleen showed no specific fluorescence.
374
R. B. Burns
DISCUSSION Because of the success of aerosol and spray vaccines in the prevention of Newcastle disease, the possible occurrence of local immunity of the respiratory tract has been postulated (Beard & Brugh, 1975). Parry & Aitken (1973) have shown that the virus neutralizing activity of respiratory tract secretions is principally associated with the lymphoid and plasma cells of the upper respiratory tract and of the paraocular and paranasal tissues of the fowl. They have shown, using class-specific antisera, that the Harderian gland plasma cells synthesize mainly IgA during the early post-infective stages of Newcastle disease. The course of immunoglobulin production in the Harderian glands of non-infected birds has been studied by Albini et al. (1973). They found that up to the 4th week after hatching most of the plasma cells produced IgM, from the 4th-9th week both IgG and IgA predominated, and later on IgA was the main immunoglobulin found. These results differ from those of Bienenstock, Gauldie & Perey (1973) who found mainly IgG and IgM with a trace amount of IgA in the Harderian glands of 5-month-old birds. Rothwell, Wight & Burns (1973) reported a predominance of IgG in the Harderian glands of nineweek-old chickens. The present agar-gel diffusion results suggest the presence of two immunoglobulin classes in the tears of BSA-stimulated birds. From the position of the precipitin lines in the plate it is suggested that they are IgA and IgG. Though there is some disagreement as to the actual immunoglobulin classes produced by the fowl Harderian gland there is general unanimity that the gland is an important lymphoid organ. Mueller et al. (1971), in a plaque assay, were able to show the production of specific haemolysins by lymphoid cells of the gland after local conjunctival immunization with sheep erythrocytes. They found antibody-producing cells in the glands of birds immunized by topical application of sheep red blood cells but not in those of birds stimulated by intravenous injection. These results are in contrast to those obtained in the present study using BSA; antibody production against BSA was seen in the glands and tears of birds immunized topically and systemically and also when the two routes were combined. The present results are in agreement with those of Mueller et al. (1971) in that there was no antibody production demonstrable in the spleen with topical application of antigen. Perhaps the differences in the two sets of results might be due to using a thymus-dependent antigen, sheep red blood cells, as opposed to a bursa-dependent antigen, BSA. Sundick et al. (1973) have presented evidence suggesting that the lymphoid cell population of the fowl Harderian gland is mainly bursa-dependent. REFERENCES AMrKEN, I.D. & PARRY, S.H. (1974) The comparative COOPER, M.D., PETERSON, R.D.A., SOUTH, M.A. & GOOD R.A. (1966) The functions of the thymus system and the serological response of the chicken, pheasant and quail to bursa system in the chicken. 3. exp. Med. 123, 75. a soluble and a particulate antigen. Immunology, 27, 623. ALBINI, B. & WICK, G. (1973) Immunoglobulin deter- MUELLER, A.P., SATO, K. & GLICK, B. (1971). The chicken lacrimal gland, gland of Harder, caecal tonsil and accesminants on the surface of chicken lymphoid cells. Int. sory spleens as sources of antibody-producing cells. Cell. Arch. Allergy, 44, 804. Immunol. 2, 140. ALBINI, B., WIcK, G., ROSE, E. & ORLANs, E. (1974) Immunoglobulin production in chicken Harderian glands. PARRY, G.H. & AITKEN, I.D. (1973). Immunoglobulin A in the respiratory tract of the chicken following exposure Int. Arch. Allergy, 47, 23. to Newcastle disease virus. Vet. Rec. 93, 258. BANG, B.G. & BANG, F.B. (1968) Localised lymphoid tissues and plasma cells in paraocular and paranasal organ sys- ROTHWELL, B., WIGHT, P.A.L. & BURNS, R.B. (1973) The Harderian glands of domestic avian species. Proc. III. tems in chickens. Amer. J. Path. 53, 735. europ. anat. Congr. Manchester, 333. BEARD, C.W. & BRUGH, M. (1975) Immunity to Newcastle SAINTE-MARIE, G. (1963) A paraffin embedding technique disease. Amer. ]. vet. Res. 36, 509. for studies employing immunofluorescence. 3. Histochem. BIENENSTOCK, J., GAULDIE, J. & PEREY, D.Y.E. (1973) Cytochem. 10, 250. Synthesis of IgG, IgA, IgM by chicken tissues: immunofluorescent and 14C amino acid incorporation studies. J. SuNDicK, R.S., ALBINI, B. & WICK, G. (1973) Chicken Harder's gland: evidence for a relatively pure bursaImmunol. 111, 112. dependent lymphoid cell population. Cell. Immunol. 7,332. COONS, A.H., LEDUC, E.H. & CoNNoLLY, J.M. (1955) Studies on antibody production. I. A method for the WIGHT, P.A.L., BURNS, R.B., ROTHWELL, B. & MACKENZIE, G.M. (1971) The Harderian gland of the domestic fowl. histochemical demonstration of specific antibody and its I. Histology, with reference to the genesis of plasma cells application to a study of the hyperimmune rabbit. J. exp. and Russell bodies. 7. Anat. 110, 307. Med. 102, 49.
Specific antibody production against a soluble antigen in the Harderian gland of the domestic fowl R. B. BURNS Anatomy Section, A.R.C. Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh
(Received 22 June 1976) SUMMARY
Three groups of chickens were immunized with bovine serum albumen (BSA). One group received the antigen i.v., the second had BSA dropped into the orbit and the third group was immunized both systemically and by topical application of BSA. Agar-gel diffusion studies showed antibodies to BSA to be present in the tears and sera of the first and third groups but only in the tears of the second group. Immunofluorescence revealed positive anti-BSA staining in lymphoid cells of the spleens of the first and third groups and in plasma cells of Harderian glands of all three groups. It is concluded that systemic and/or topical applications of BSA affect the tears and Harderian gland whereas only systemic injections produce an effect in the spleen and serum. The Harderian gland may have an important part to play in the local immunologic mechanism of the eye and upper respiratory tract of the fowl.
INTRODUCTION Harder's gland is the dominant orbital gland in the domestic fowl (Wight et al., 1971). Though its main functions are to lubricate the movement of the nictitating membrane, wet the surface of the eyeball and nourish the avascular cornea, it probably plays a part in the local immunity of the eye and upper respiratory tract since it has a large age-dependent population of plasma cells (Bang & Bang, 1968; Wight et aL, 1971), surface immunoglobulin-bearing cells (Albini & Wick, 1973) and some antibodyproducing cells (Mueller, Sato & Glick, 1971). Sundick, Albini & Wick (1973) have shown that the lymphoid cells in the gland are mainly bursa-dependent suggesting that the gland is active in humoral, as opposed to cellular, antibody responses (Cooper et al., 1966). The present experiments study the effects of conjunctival and systemic immunizations with bovine serum albumen on the Harderian gland and tears of the domestic fowl.
MATERIALS AND METHODS Animals. Nine-week-old White Leghorn chickens were used. Immunization. Eighteen birds were divided into three groups of six. Group A birds received an i.v. injection of 0 5 ml 300 bovine serum albumen (BSA) in saline twice a week for 2 weeks. Group B received eye drop instillations (0.5 ml into each eye) of 3%o BSA twice a week for two weeks and group C received similar amounts of 3% BSA both i.v. and dropped into
the orbit twice a week for 2 weeks. Untreated and saline-treated birds served as controls. A week after the final injections and orbital applications of BSA the birds were bled from the brachial vein to obtain serum and tears were collected. Tear collection. Lachrymation was induced by dropping glycerol onto the eyeball. This immediately caused tears to well up into the pocket formed by gently pinching the lower eyelid. About 0 5 ml of fluid was collected from each eye using a Pasteur pipette. About two thirds of the fluid collected, as estimated by subtracting the glycerol used from the total volume of fluid, appeared to be composed of tears. Ouchterlony Plate Analyses. Antibodies against BSA were detected by double diffusion in agar gel (Ionagar No. 2, Oxoid) using 8%NaCl in the agar (Aitken & Parry, 1974). Neat serum and undiluted tear-glycerol mixtures were used in the
1%y
Correspondence: Mr R. B. Burns, Mains Road, Edinburgh EH9 8JF.
Agricultural Research Council's Poultry Research Centre, King's Buildings, West
371
372
R. B. Burns
peripheral wells and 1% BSA in phosphate-buffered saline in the central well. Agar plates were stained with naphthalene black. Immunofluorescence. Immediately after the blood and tear collections the birds were killed with pentobarbitone sodium ('Nembutal', Abbott Laboratories). The Harderian glands and spleen were removed, fixed in cold 95% ethanol and waxembedded according to the method of Sainte-Marie (1963). Wax sections, 4 ,um thick, were cut, mounted on glass slides and stained for specific histochemical demonstration of antibody using the method of Coons, Leduc & Connolly (1955). Dewaxed sections were flooded with 1% BSA in saline and incubated at room temperature for 30 min in a humid atmosphere. After washing in phosphate-buffered saline (PBS) the sections were incubated with fluorescein-conjugated rabbit anti-.BSA antiserum (Nordic Pharmaceuticals) diluted 1:10 with PBS for 30 min. The sections were again washed in PBS and then mounted in buffered glycerol. Control sections were prepared in a similar manner except that the antigen layer was omitted. Preparations were examined using a microscope fitted with a mercury vapour lamp. Fluorescence photographs were taken on Ilford HPA film with a constant exposure of 60 sec.
RESULTS The presence of antibody against BSA in tears and serum was demonstrated by agar-gel diffusion. Table 1 summarizes the results. The serum from all six birds in group A showed a positive reaction against BSA in the agar plates whereas only three out of six birds had antibodies against BSA in their tears. Four out of six birds in group B had antibodies against BSA in their tears though none of the sera in this group showed a positive reaction against BSA. The results of group C tears and sera were similar to those of group A with all six sera having antibodies against BSA and four out of the six tear collections being positive. When serum reacted against BSA in Ouchterlony plates a single precipitin line was formed (Fig. 1) whereas when tears showed antibodies against BSA two precipitin lines formed (Fig. 1). Because the TABLE 1. Ouchterlony plate analyses of sera and tears from BSA-stimulated birds
Group A Group B Group C
Number of birds
Number of birds
Route of immunization
showing positive sera
showing positive tears
i.v. Injections Ocular drops i.v. Injections and ocular drops
6/6 0/6 6/6
3/6 4/6 4/6
FiG. 1. Double-diffusion test in agar gel. Bovine serum albumen (BSA) in the centre well with serum (S) and tears (T) from a bird immunized by eyedrop and i.v. injection of BSA in the side wells. Stained with naphthalene black.
Fowl Harderian gland antibody production 373 inner of the two precipitin lines produced by the tears gave a reaction of identity with the single line given by the serum it is suggested that in the serum an IgG response has taken place whereas in the tears both IgG and IgA are present as antibodies. When sections of Harderian gland and spleen were stained by the sandwich technique for anti-BSA fluorescence, anti-BSA activity was seen in the glands of all three experimental groups. Fluorescence was confined to plasma cells (Fig. 2). It was usual for the whole cytoplasm of the cell to fluoresce a bright apple-green colour (Fig. 2) though occasionally plasma cells with brightly fluorescing spots (immunoglobulin surface determinants) were also seen (Fig. 3).
FIG. 2. Harderian gland plasma cells showing specific fluorescence for anti-BSA antibody in a bird immunized by eye-drop applications of BSA. (Magnification x 384.)
FIG. 3. Harderian gland plasma cells (arrowed) showing fluorescent spots specific for anti-BSA antibody in a bird immunized by eyedrop and i.v. injection of BSA. (Magnification x 384.)
Anti-BSA fluorescing plasma cells in the Harderian glands occurred either singly or in groups of four or five (Figs 2 and 3). Occasionally the tracts between epithelial cells lining the primary and secondary tubules showed some specific fluorescence. Positive anti-BSA fluorescence was seen in lymphoid cells of spleens of group A and C birds but not in group B. Control sections of Harderian gland and spleen showed no specific fluorescence.
374
R. B. Burns
DISCUSSION Because of the success of aerosol and spray vaccines in the prevention of Newcastle disease, the possible occurrence of local immunity of the respiratory tract has been postulated (Beard & Brugh, 1975). Parry & Aitken (1973) have shown that the virus neutralizing activity of respiratory tract secretions is principally associated with the lymphoid and plasma cells of the upper respiratory tract and of the paraocular and paranasal tissues of the fowl. They have shown, using class-specific antisera, that the Harderian gland plasma cells synthesize mainly IgA during the early post-infective stages of Newcastle disease. The course of immunoglobulin production in the Harderian glands of non-infected birds has been studied by Albini et al. (1973). They found that up to the 4th week after hatching most of the plasma cells produced IgM, from the 4th-9th week both IgG and IgA predominated, and later on IgA was the main immunoglobulin found. These results differ from those of Bienenstock, Gauldie & Perey (1973) who found mainly IgG and IgM with a trace amount of IgA in the Harderian glands of 5-month-old birds. Rothwell, Wight & Burns (1973) reported a predominance of IgG in the Harderian glands of nineweek-old chickens. The present agar-gel diffusion results suggest the presence of two immunoglobulin classes in the tears of BSA-stimulated birds. From the position of the precipitin lines in the plate it is suggested that they are IgA and IgG. Though there is some disagreement as to the actual immunoglobulin classes produced by the fowl Harderian gland there is general unanimity that the gland is an important lymphoid organ. Mueller et al. (1971), in a plaque assay, were able to show the production of specific haemolysins by lymphoid cells of the gland after local conjunctival immunization with sheep erythrocytes. They found antibody-producing cells in the glands of birds immunized by topical application of sheep red blood cells but not in those of birds stimulated by intravenous injection. These results are in contrast to those obtained in the present study using BSA; antibody production against BSA was seen in the glands and tears of birds immunized topically and systemically and also when the two routes were combined. The present results are in agreement with those of Mueller et al. (1971) in that there was no antibody production demonstrable in the spleen with topical application of antigen. Perhaps the differences in the two sets of results might be due to using a thymus-dependent antigen, sheep red blood cells, as opposed to a bursa-dependent antigen, BSA. Sundick et al. (1973) have presented evidence suggesting that the lymphoid cell population of the fowl Harderian gland is mainly bursa-dependent. REFERENCES AMrKEN, I.D. & PARRY, S.H. (1974) The comparative COOPER, M.D., PETERSON, R.D.A., SOUTH, M.A. & GOOD R.A. (1966) The functions of the thymus system and the serological response of the chicken, pheasant and quail to bursa system in the chicken. 3. exp. Med. 123, 75. a soluble and a particulate antigen. Immunology, 27, 623. ALBINI, B. & WICK, G. (1973) Immunoglobulin deter- MUELLER, A.P., SATO, K. & GLICK, B. (1971). The chicken lacrimal gland, gland of Harder, caecal tonsil and accesminants on the surface of chicken lymphoid cells. Int. sory spleens as sources of antibody-producing cells. Cell. Arch. Allergy, 44, 804. Immunol. 2, 140. ALBINI, B., WIcK, G., ROSE, E. & ORLANs, E. (1974) Immunoglobulin production in chicken Harderian glands. PARRY, G.H. & AITKEN, I.D. (1973). Immunoglobulin A in the respiratory tract of the chicken following exposure Int. Arch. Allergy, 47, 23. to Newcastle disease virus. Vet. Rec. 93, 258. BANG, B.G. & BANG, F.B. (1968) Localised lymphoid tissues and plasma cells in paraocular and paranasal organ sys- ROTHWELL, B., WIGHT, P.A.L. & BURNS, R.B. (1973) The Harderian glands of domestic avian species. Proc. III. tems in chickens. Amer. J. Path. 53, 735. europ. anat. Congr. Manchester, 333. BEARD, C.W. & BRUGH, M. (1975) Immunity to Newcastle SAINTE-MARIE, G. (1963) A paraffin embedding technique disease. Amer. ]. vet. Res. 36, 509. for studies employing immunofluorescence. 3. Histochem. BIENENSTOCK, J., GAULDIE, J. & PEREY, D.Y.E. (1973) Cytochem. 10, 250. Synthesis of IgG, IgA, IgM by chicken tissues: immunofluorescent and 14C amino acid incorporation studies. J. SuNDicK, R.S., ALBINI, B. & WICK, G. (1973) Chicken Harder's gland: evidence for a relatively pure bursaImmunol. 111, 112. dependent lymphoid cell population. Cell. Immunol. 7,332. COONS, A.H., LEDUC, E.H. & CoNNoLLY, J.M. (1955) Studies on antibody production. I. A method for the WIGHT, P.A.L., BURNS, R.B., ROTHWELL, B. & MACKENZIE, G.M. (1971) The Harderian gland of the domestic fowl. histochemical demonstration of specific antibody and its I. Histology, with reference to the genesis of plasma cells application to a study of the hyperimmune rabbit. J. exp. and Russell bodies. 7. Anat. 110, 307. Med. 102, 49.
